Cadmium mediates pyroptosis of human dermal lymphatic endothelial cells in a NLRP3 inflammasome-dependent manner

Abstract

Pyroptosis is a form of inflammasome-trigged programmed cell death in response to a variety of stimulators, including environmental cytotoxic pollutant Cadmium (Cd). Vascular endothelial cell is one of the first-line cell types of Cd cell toxicity. Studies report that Cd exposure causes pyroptosis in vascular endothelial cells. Vascular and lymphatic endothelial cells have many common properties, but these two cell types are distinguished in gene expression profile and the responsive behaviors to chemokine or physical stimulations. Whether Cd exposure also causes pyroptosis in lymphatic endothelial cells has not been investigated. Here, we found that Cd treatment significantly decreased the viability of human dermal lymphatic endothelial cells (HDLECs). Cd treatment induced inflammasome activation indicated by elevated cleavage of pro-caspase-1 into active form Casp1p20, elevated secretion of pro-inflammatory cytokines and production of reactive oxygen species (ROS). Flow cytometry showed that caspase-1 activity was significantly increased in Cd-treated cells. Moreover, knockdown of NLRP3 effectively rescued Cd-induced inflammasome activation and pyroptosis in HDLECs. Collectively, our results indicated that Cd induced pyroptosis in a NLRP3 inflammasome-dependent manner in lymphatic endothelial cells.

INTRODUCTION

Cadmium (Cd), a metallothionein-binding divalent cationt with a long biological half-life, can easily accumulate in animal tissues or organs (Hamid et al., 2019; Waalkes, 2003). Cd is included in the first group of human carcinogens (Wang et al., 2020b). Cd exposure is associated with damage to multiple organs, including kidney, liver, lung, brain, nerve, bone and the cardiovascular system (Wang et al., 2018; Dong et al., 2014; Chen et al., 2016a). Excessive intake and insufficient excretion of cadmium have become a global problem as industrialization deepens (Liu et al., 2015; Dong et al., 2014). Fertilizer application, sewage irrigation, mining, smelting and fuel combustion are the main sources of Cd pollution. Cd is absorbed by the digestive tract and lungs, and circulates in the blood and lymphatic systems before entering into tissues or organs (Templeton and Liu, 2010). During this process, Cd directly interacts with the innermost layer of the circulatory system, and causes significant adverse effects on the endothelial cells.

Pyroptosis is a form of programmed cell death associated with pro-inflammatory signals. Cells undergoing pyroptosis exhibit cell swelling and lysis, chromatin fragmentation and cell membrane pore formation, followed by the release of intracellular proinflammatory cytokines (Long et al., 2020). The canonical activation of pyroptosis is executed through inflammasome cascade (Yu et al., 2021). NLRP3 inflammasome, which is composed of pattern recognition receptor (PRR), adapter protein apoptosis-associated speck-like protein (ASC), and caspase-1, is the most well-known inflammasome type (Wang et al., 2019). The NLRP3 inflammasome can be activated by pathogen associated molecular patterns (PAMPs), endogenous danger associated molecular patterns (DAMPs), such as pore-forming toxins, reactive oxygen species (ROS), ATP (Yu et al., 2020; He et al., 2016) and Cd (Chen et al., 2016a). Upon the activation of inflammasome, caspase-1 is recruited by the NLRP3 complex and autocatalyzed into the active form consisting of two 20-kD fragments (Casp1p20) and two 10-kD fragments (Hu et al., 2018). The active caspase-1 complex cleaves GSDMD protein and mediates the formation of pyroptotic cell membrane pores. In addition, the active caspase-1 complex also participates in maturation of the IL-18 and IL-1β precursors (Cromer et al., 2014; Fang et al., 2020), which further strengthens the pro-inflammatory and pyroptotic status.

The lymphatic system is a complement to the blood system, and plays an integral role in regulating interstitial fluid environmental balance, immune cell transport and dietary fat absorption (Alitalo, 2011; Henderson et al., 2020; von der Weid, 2019; Martel et al., 2013). Lymphatic vessels are blind lumens, and absorb stromal fluid for re-circulation (García-Caballero et al., 2016). Studies show that Cd exposure causes pathological reactions in the lymphatic system. In mice models, footpad injection of CdCl₂ induces hyper activation and proliferation of T- and B-cells in popliteal lymph nodes (Carey et al., 2006). In Itai-itai disease, the most severe form of chronic cadmium poisoning, nephrotic edema develops with dilated lymphatic vessel (Imura et al., 2019). However, the evidence concerning the biological functions and related molecular mechanisms of Cd in lymphatic endothelial cells is still very limited.

Lymphatic and vascular endothelial cells have many common properties in morphology, structure, function and gene expression (Yuan et al., 2019). However, there are still a cohort of remarkable distinctions between lymphatic and vascular endothelial cells. Compared with vascular endothelial cells, lymphatic endothelial cells form wider lumen, characterized by collapsed lumen wall and overlapping intercellular junctions, directly connected to the interstitial collagen via anchoring filaments, as a result of full or partial lack of basement membrane, and are not invested by pericytes (Pepper and Skobe, 2003). Lymphatic endothelial cells also have a unique gene expression profile, with high expression of LYVE-1, not expressed in vascular endothelial cells, and lack of expression of CD34, a common vascular endothelial cell marker (Podgrabinska et al., 2002). High throughput analysis shows that the differentially expressed genes in lymphatic endothelial cells, such as rab GTPases, sec-related proteins, vesicle associated membrane proteins and ATPases, are functionally implicated in protein sorting and trafficking (Podgrabinska et al., 2002). Most importantly, lymphatic endothelial cells also exhibit a distinct pattern in response to chemokine or physical stimulations. Lymphatic endothelial cells have a lower responsive sensitivity upon TNF-α stimulation, manifested by lower fold increase of VCAM-1, ICAM-1 and E-selectin expression (Sawa et al., 2007). Lymphatic endothelial cells appear to be more vulnerable to air pollutant PM2.5-induced cell death than vascular endothelial cells (Cui et al., 2020). Cd has been found to activate NLRP3 inflammasomes, the secretion of proinflammatory cytokines, and subsequent pyroptosis in vascular endothelial cells (Chen et al., 2016a). Whether this mechanism occurs in lymphatic endothelial cells has not been investigated.

The purpose of this study was to explore the functions of Cd in the pyroptosis of HDLECs. We evaluated the cytotoxic effect of Cd exposure on HDLECs and elucidated its roles of NLRP3 inflammasome activation, the secretion of pro-inflammatory cytokines and the formation of cell membrane pores during pyroptosis. This study provides a potential therapeutic strategy for the treatment of diseases related to lymphatic endothelial cell pyroptosis.

MATERIALS AND METHODS

Cell culture

HDLECs were purchased from Bai Li Biology (Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (Manassas, VA, USA). Human cardiac microvascular endothelial cells (HCMECs) were purchased from Zhongqiaoxinzhou Biotechnology (Shanghai, China). HDLECs, HUVECs and HCMECs were cultured using an endothelial cell medium kit (ScienCell, San Diego, CA, USA). Cells were incubated under a 37°C and 5% CO₂ atmosphere.

Cell viability assay

Cell viability was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazo-lium bromide (MTT) (Solarbio, Beijing, China) assay or CCK-8 assay (APExBIO, Houston, TX, USA) according to the manufacturer’s instructions. Cells were treated with different concentrations of CdCl₂ (Sigma Aldrich, St. Louis, MO, USA) dissolved in phosphate-buffered saline for 24 hr, and co-cultured with 10 μL 5 mg/mL MTT solution or 1/10 volume CCK-8 reagent for 4 and 2 hr, respectively. Dimethyl sulfoxide (DMSO) was added to dissolve aggregates following medium aspiration. The culture was shanked carefully, and the absorbance was measured at 490 nm for MTT assay and 450 nm for CCK-8 assay.

Intracellular ROS measurement

The production of intracellular ROS was measured using a Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocols. DCFH-DA was diluted with the serum-free medium at 1:1000 to achieve a final concentration of 10 μM. Cell culture medium was removed and the diluted DCFH-DA was added into cell culture. Cells were incubated at 37°C for 20 min, and washed three times in a serum-free cell culture medium. The fluorescence was observed under a fluorescent microscope.

Quantitative RT-PCR (qRT-PCR) analysis

Total RNA was isolated by RNAiso Plus reagent and reverse-transcribed into cDNA using HiScript III RT SuperMix (Vazyme, Nanjing, China) following the manufacturer’s instructions. qRT-PCR was performed using ChamQ SYBR qPCR Master Mix reagent (Vazyme). Primer sequences were as follows: interleukin-1β (IL-1β): forward 5´-CAC GAT GCA CCT GTA CGA TCA-3´, reverse 5´-GTT GCT CCA TAT CCT GTC CCT-3´; Tumor necrosis factor-α (TNF-α): forward 5´-GTG ATC GGC CCC CAG AGGGA-3´, reverse 5´-CAC GCC ATT GGC CAG GAG GG-3´; Monocyte chemoattractant protein 1 (MCP-1): forward 5´-GGA CCA CCT GGA CAA GCA AA-3´, reverse 5´-GGT GTC TGG GGA AAG CTA GG-3´; IL-6: forward 5´-CCA CTC ACC TCT TCA GAA C-3´, reverse 5´-CTT TGC TGC TTT CAC ACA T-3´; IL-8: forward 5´-GCA GAG GGT TGT GGA GAA-3´, reverse 5´-ACT GGC ATC TTC ACT GAT TC-3´; GAPDH: forward 5´-TTT GGC TAC AGC AAC AGG GT-3´, reverse 5´-GGG AGA TTC AGT GTG GTG GG-3´; AIM2: forward 5´- TTGAGACCCAAGAAGGCAAG-3´; reverse 5´-CGTGAGGCGCTATTTACCTC´.

Western Blot analysis

HDLECs were washed twice with phosphate-buffered saline and then lysed in RIPA buffer supplemented with PMSF (1 mM). Proteins were separated on SDS-PAGE gels and transferred onto the PVDF membrane (Immobilon, Massachusetts, USA). Membranes were blocked with 5% non-fat dry milk for 2 hr at room temperature and incubated with primary antibodies overnight at 4°C. Primary antibodies were as follows: rabbit anti-NLRP3 antibody (Proteintech, Wuhan, China, 1:1000), rabbit anti-caspase-1 antibody (Proteintech, 1:1000), rabbit anti-Casp1p20 antibody (Proteintech, 1:1000), mouse anti-Pro-IL-1β antibody (CST, Danvers, MA, USA; 1:1000) and mouse anti-IL-1β antibody (CST, 1:1000), Immunoreactivity was visualized with HRP-linked secondary antibodies (Millipore, Billerica, MA, USA) under a chemiluminometer.

siRNA transfection

The NLRP3 and AIM2 siRNA kit were purchased from Jishi Biotech (NLRP3 siRNA_1 and AIM2 siRNA; Beijing, China) or RiboBio (NLRP3 siRNA_2; Guangzhou, China). HDLECs were transfected with 100 nm NLRP3 siRNA, AIM2 siRNA or a non-specific siRNA using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Cells were used for analysis 24 hr after transfection. The siRNA sequences (sense) were: NLRP3 siRNA_1-5’GCUUUGUCCUCGGUACUCA3’, NLRP3 siRNA_2-5’GAGAGACCUUUAUGAGAAA3’, AIM2 siRNA-5’GGAACAAUUGUGAAUGGUU3’.

Flow cytometry

Caspase-1 activity was detected by FLICA 660 Caspase-1 Assay (Immunochemistry, Lumington, MN, USA) according to the manufacturer’s protocol. Green Live/Dead Stain (Immunochemistry) was used to mark cells with membrane pore formation. Collect 295 μL cells of a density of 4 × 10⁵ cells/mL density and supplement with 5 μL FLICA 660 working fluid. Cells were cultured at 37°C for 1 hr, and then washed with a cell wash buffer to remove the unbound FLICA reagent. Cells were then stained with 1 μM Green Live/Dead Stain for 10 min at 37°C. Cell staining was analyzed on a flow cytometer.

Propidium Iodide (PI) staining

To confirm cell membrane pore formation, we applied the cell apoptosis and necrosis Assay kit (Beyotime). Cells were stained with PI (5 ng/mL) and Hoechst33342 (5 ng/mL) 30 min at 37°C protected from light, and visualized under a fluorescent microscope.

ELISA

IL-6 and TNF-α kit were purchased from Beyotime Biotechnology. ELISA measurement was performed according to the manufacturer’s protocols. Briefly, cell culture medium was collected and centrifuged at 10,000 × g at 4°C for 10 min to remove cell debris, and 100 μL supernatant was added into antibody-coated 96-well plate wells. The plate was incubated at room temperature for 2 hr and washed for 5 times. The captured proteins were bounded with biotinylated specific antibodies and further labeled with Streptavidin-HRP. Add TMB solution and incubate at room temperature for 30 min. The absorbance at 450 nm was measured using a microplate reader (Winooski, VT, USA).

RESULTS

Cd induced cell cytotoxicity in HDLECs

To determine the cytotoxic effects of Cd on lymphatic endothelial cells, we performed MTT and CCK8 assay in HDLECs treated with 0, 1, 5, 10, 50 and 100 μM CdCl₂ for 24 hr. MTT assay showed that HDLEC viability was slightly increased by 1 μM Cd treatment (Fig. 1A), consistent with our results that low dosage Cd treatment increased cell viability (Chen et al., 2016b). However, treatment of 100 μM Cd significantly decreased HDLEC viability (Fig. 1A). Similar results were achieved using CCK-8 methods, although 5 μM and 10 μM Cd also increase HDLEC viability (Fig. 1B). To investigate the responsive differences between vascular and endothelial cells, we introduced endothelial cells HCMECs and HUVECs into study. The EC50 of HDLECs was compatible to HUVECs, but much lower than that of HCMECs (Fig. S1). To exclude the influence of fetal bovine serum on cell viability, HDLECs were serum-starved for 24 hr and subsequently treated with dosages of Cd concentration. The results of CCK-8 assay showed that the sensitivity of HDLECs to Cd treatment did not change significantly after serum starvation (Fig. S2). Pyroptosis is a subtype of programmed cell death with significant cytotoxicity. Based on these results, HDLECs were treated with 100 μM Cd for 24 hr in the following studies. We used a Reactive Oxygen Species Assay Kit to detect whether Cd could increase the production of ROS. As shown in Fig. 1C and D, the level of ROS was significantly increased after HDLECs were treated with 100 μM CdCl₂ (P < 0.05). These results showed that Cd is cytotoxic to lymphatic endothelial cells.

Fig. 1

Cd induces cell cytotoxicity in HDLECs. HDLECs were treated with different concentrations of Cd for 24 hr. Cell viability was determined by (A) MTT and (B) CCK8 assay. The data represent the mean ± standard deviation. n = 3. (C) Representative images of cell staining after intracellular ROS assay with the treatment of 100 μΜ Cd after 24 hr. (D) Bar Graph of intracellular reactive oxygen species by immunofluorescent staining. The data are presented as the mean ± standard error of the mean. n = 3. n.s., non-significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Cd increased the production of pro-inflammatory cytokines in HDLECs

The activation of pyroptosis is accompanied by the release of pro-inflammatory cytokines, one of the main characteristics that is distinguished from other types of programmed cell death. The mRNA level of pro-inflammatory cytokines in HDLECs, including IL-1β (Fig. 2A), IL-6 (Fig. 2B), IL-8 (Fig. 2C), TNF-α (Fig. 2C) and MCP-1 (Fig. 2D) were all significantly increased by 100 μM Cd treatment. Particularly, the mRNA level of IL-8 and IL-6 in Cd-treated HDLECs were remarkably up-regulated by > 20 and > 6 folds, respectively. ELISA further demonstrated that Cd treatment could increase the secretion of TNF-α (Fig. S3A) and IL-6 (Fig. S3B) in the culture supernatant. In summary, our results showed that the expression of pro-inflammatory cytokines was significantly increased in HDLECs treated with Cd.

Fig. 2

The effect of Cd on proinflammatory cytokine expression in HDLECs. HDLECs were exposed to the concentration of 100 μM Cd for 24 hr. (A) qRT-PCR analyses of IL-1β mRNA expression in HDLECs. (B) qRT-PCR analyses of IL-6 mRNA expression in HDLECs. (C) qRT-PCR analyses of IL-8 mRNA expression in HDLECs. (D) qRT-PCR analyses of TNF-α mRNA expression in HDLECs. (E) qRT-PCR analyses of MCP-1 mRNA expression in HDLECs. The data are presented as the mean ± standard deviation. n = 3; *, P < 0.05; ***, P < 0.001.

Cd activates NLRP3 inflammasome and causes pyroptosis in HDLECs

NLRP3 inflammasome activation plays an important role in pyroptosis. To evaluate the influence of NLRP3 inflammasomes on the pyroptosis of lymphatic endothelial cells, we monitored the activation of NLRP3 activation in Cd-treated HDLECs. Western Blot results showed that the protein level of NLRP3 was significantly increased by 100 μM Cd treatment. The cleavage of IL-1β and caspase-1 is considered an indicator of NLRP3 inflammasome activation. We found that the protein levels of Casp1p20 (Fig. 3A) and mature IL-1β (Fig. 3B) were both significantly increased in HDLECs with Cd treatment. Flow cytometry analysis showed that Caspase-1 activity was very weak in healthy HDLECs with a positive-staining percentage of 1.1%, and 100 μM Cd treatment could increase the positive percentage to 56.0% (Fig. 3C and D). The number of HDLECs with membrane pores was also increased by more than 40 fold in the 100 μM Cd-treated group (Fig. 3E and F). These studies showed that Cd treatment elicited pyroptosis in HDLECs.

Fig. 3

Cd induces pyroptosis in HDLECs. (A) Representative immunoblots of NLRP3, Caspase1 and cleaved Caspase1 (Casp1p20) in HDLECs with or without the treatment of 100 μM Cd for 24 hr. (B) Representative immunoblots of Pro-IL-1β and IL-1β in HDLECs with or without the treatment of 100 μM Cd for 24 hr. (C) Representative image of Membrane pore formation (Green/Dead staining, horizontal) and caspase-1 activation (FLICA 660-YVAD-Caspase1 staining, vertical) by flow cytometry Analysis. (D) Bar Graph of Double positive staining for Green/Dead staining Caspase1 staining. The data are presented as the mean ± standard error of the mean. n = 3. (E) Immunofluorescent staining of Hoechst33342 (blue) and PI (red) in HDLECs treated with or without 100 μM Cd for 24 hr. (F) Bar Graph of PI positive cell of total cells. The data are presented as the mean ± standard deviation. n = 3. **, P < 0.01.

Cd induces pyroptosis through NLRP3 inflammasome

We tested whether Cd-induced pyroptosis in HDLECs was mediated through the NLRP3 inflammasome pathway. For this purpose, HDLECs were transfected with NLRP3 siRNA to knock down NLRP3 expression (Fig. 4A). Knockdown of NLRP3 remarkably decreased Casp1p20 protein level (Fig. 4A; Fig. S4A), and had no detectable effects on the percentage of HDLECs with membrane pores (PI positive) (Fig. 4B and C; Fig. S4B and C). Consistent with the aforementioned results, Cd treatment markedly increased the percentage of cells with membrane pores, which could be significantly rescued by NLRP3 knockdown (Fig. 4B and C; Fig. S4B and C). To test whether another regular inflammasome, AIM2 inflammasome, participates in Cd-mediated HDLEC pyroptosis, we knocked down the expression of AIM2 in HDLECs (Fig. S5A). PI staining showed that knockdown of AIM2 had no significant influence on HDLEC membrane pore formation, and could not significantly rescue Cd-induced membrane pore formation (Fig. S5B and C). These results indicated that Cd-induced pyroptosis of HDLECs was dependent on NLRP3 inflammasome.

Fig. 4

Cd induces cell pyroptosis though activating NLRP3 inflammasomes. HDLECs were transfected with NLRP3-targeting siRNA or control non-specific siRNA for 24 hr. (A) Representative immunoblots of NLRP3 and cleaved Caspase-1 (Casp1p20) in HDLECs with or without the treatment 100 μM Cd for 24 hr. (B) The transfected cells were then cultured for another 24 hr in the presence or absence of Cd. Immunofluorescent staining of Hoechst33342 (blue) and PI (red) in HDLECs transfected NLRP3-targeting siRNA or a control non-specific siRNA. (C) Bar Graph of PI positive cell of total cells. The data are presented as the mean ± standard deviation. n = 3; n.s., non-significant; *, P < 0.05.

DISCUSSION

In this study, we found that Cd induced cytotoxicity in lymphatic endothelial cells, which was at least partially mediated through pyroptosis. Cd exposure elicited pyroptosis in lymphatic endothelial cells, evidenced by increased cleavage of Caspase-1 and IL-1β, ROS production, percentage of Caspase-1 positive HDLECs, expression and secretion of pro-inflammatory cytokines and pore formation cell membrane. Further experiments showed that Cd-induced pyroptosis was mediated through NLRP3 inflammasome rather than AIM2 inflammasome. Our study provided a theoretical basis of the biological roles of Cd in lymphatic endothelial cells. Moreover, we found that lymphatic endothelial cells seemed to be more sensitive to Cd induced pyroptosis than vascular endothelial cells (HDLECs VS HUVECs) (Chen et al., 2016a), consistent with a previous study reported that lymphatic endothelial cells were more vulnerable to another environmental pollutant PM2.5 than vascular endothelial cells (Cui et al., 2020). However, supporting evidence comparing the resistance or sensitivity of lymphatic and vascular endothelial cells to Cd stimulation is still very limited, and more efforts shall be made to clarify this.

Cd is one of the major heavy metals that threatens human health. As a substance from a wide range of sources, Cd has several toxic effects on cells, including abnormal proliferation and apoptosis, and tumorigenic effects (Lee et al., 2020). Cd can directly or indirectly act on vascular endothelial cells, resulting in the destruction of the junction structure between cells and increase of endothelial barrier permeability (Dong et al., 2014; Hu et al., 2016; Li et al., 2016). Consistent with this, Karmakar et al. (2020) report that increased permeability of cell membrane is found in the process of cell pyroptosis. Therefore, this implies that the cell membrane pore formation as a result of pyroptosis is an important aspect in Cd-mediated cytotoxicity in HDLECs. In our study, MTT and CCK-8 assay demonstrated that Cd had significant cytotoxicity and significantly reduced the viability of HDLECs. ROS, a subset of bioactive molecules that cause cell injury and death, is one of the main DAMPs involving in Cd-mediated pyroptosis (He et al., 2016; Yu et al., 2020). Studies show that ROS induces pyroptosis by activating inflammasome pathway in vascular endothelial cells (Long et al., 2020). Cd-mediated ROS damage mechanism is involved in Cd toxicity to kidney, liver, spleen, spleen and the immune system (Zhang et al., 2021; Tang et al., 2019). Here, we also found that Cd increased ROS production in Cd-treated lymphatic endothelial cells, consistent with previous studies in vascular endothelial cells.

Pyroptosis is a programmed cell death pathway triggered by inflammasome activation. The activation of inflammasome plays a central role in the progress of pyroptotic procedure. NLRP3 inflammasome is the most common inflammasome subtype and can be activated by a variety of substances, including Cd (Rodrigues et al., 2021), nicotine (Wu et al., 2018) and acrolein (Jiang et al., 2018a). In this study, we also found that Cd induced pyroptosis through NLRP3 inflammasome, indicated by increased NLRP3 protein level, Casp1p20 production, and IL-1β expression, maturation and secretion. The formation of cell membrane pores, the hallmark of pyroptosis, was also dramatically increased in Cd-treated HDLECs. The increase of Caspase-1 activity in live HDLECs treated with Cd further confirmed that Cd activates NLRP3 inflammasomes and induces pyroptosis of HDLECs.

Inflammasome is activated in response to infection or external substances (Choi et al., 2019; Wang et al., 2019). The major inflammasome types, including NLRP1 (Tan et al., 2014), AIM2 (Gao et al., 2019) and NLRP3 inflammasomes, all are reported to induce cell pyroptosis. NLRP3 inflammasomes play a primary role in the activation of pyroptosis (Xue et al., 2019). NLRP3 proteins self-oligomerize and recruit ASC proteins to trigger inflammasome assembly and promote a powerful immune response (Song and Li, 2018). Activation of caspase-1 through the NLRP3 inflammasome leads to cleavage of GSDMD and production of porous GSDMD-N, which results in the formation of cell membrane pore (Wang et al., 2020a). In the present study,, we demonstrated the expression level of Casp1p20 protein was decreased in HDLECs with NLRP3 knockdown in the presence of Cd. Knocked-down HDLECs also had a higher cell membrane integrity in Cd-treated HDLECs. In context with aforementioned conclusion, Cd activated pyroptosis of lymphatic cells in a NLRP3-dependent manner.

Inflammation is the basis of many diseases and plays an integral role in the development of various diseases. Many studies have shown that Cd can induce the production of pro-inflammatory cytokines in a variety of cells and tissues, including vascular endothelial cells and tissue cells (Hossein-Khannazer et al., 2020). Recent studies have also shown that Casp1p20 acts on pro-IL-1β and pro-IL-18 and catalyzes their extracellular secretion into biologically active forms (Choi et al., 2019; Jiang et al., 2018a; Xi et al., 2016). Further, the formation of cell membrane pores during pyroptosis facilitates the release of cell contents into the tissue fluid, including a large number of pro-inflammatory cytokines (DiPeso et al., 2017). In this study, we found that the expression level of IL-1β, IL-6, IL-8, TNF-α, and MCP-1 were significantly increased after Cd exposure in HDLECs. Therefore, we speculate that the pyroptosis of HDLECs may be one of the mechanisms by which Cd induces the pro-inflammatory response of cells and promotes the pathophysiological progression of diseases such as atherosclerosis and lymphedema. Therefore, anti-inflammation therapy could be a potential strategy to treat pyroptosis-related diseases.

In recent years, the importance of the lymphatic system in tumor metastasis, chronic inflammation, and other pathological states has been recognized (Alitalo, 2011). Lymphedema is an inflammation-related disorder in which lymph fluid is disrupted and fails to circulate properly back into the blood system (Bertelli et al., 2020). Low-grade inflammation in the state of lymphedema is a contributing factor to lymphatic dysfunction (von der Weid, 2019). The expression of pro-inflammatory cytokines increased in lymphedema, accompanied by pathological manifestations such as tissue fluid accumulation, lymphatic fibrosis, and adipose tissue deposition (Jiang et al., 2018b). Cd has been reported to induce inflammation (Zhong et al., 2017; Hossein-Khannazer et al., 2020). This further indicates that HDLECs may produce an inflammatory cell survival environment and promote lymphedema after Cd exposure. Therefore, clarifying the relationship between lymphedema and Cd-induced pyroptosis will provide a new strategy for the prevention and diagnosis of the disease.

Our results show that treatment of lymphatic endothelial cells with Cd resulted in NLRP3 inflammasome activation and pyroptosis, indicated by increased NLRP3 protein expression, caspase-1 cleavage, IL-1β and IL-18 production, and the impairment of cell membrane integrity. We demonstrated that the activation of Cd-induced pyroptosis of lymphatic endothelial cells is dependent on NLRP3 inflammasomes.

ACKNOWLEDGMENTS

This study is supported by grants from Department of Science and Technology of Shandong Province (2019GSF108234), National Natural Science Foundation of China (81873473, 91939110, 81700253), Academic Promotion Program of Shandong First Medical University (2019QL014) and Shandong Taishan Scholarship (JL).

Conflict of interest

The authors declare that there is no conflict of interest.

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